Field of the Invention
The present teachings generally relate to compositions, processes, methods, and kits for preparation of samples containing genetic material for downstream detection and/or quantitation analysis.
Description of the Related Art
Laser capture microdissection (LCM) is a technique for selecting or micro dissecting specific cell(s) from a mixed population, usually under microscopic visualization. LCM techniques are used to isolate a single cell or few cells from fresh or archived formalin-fixed paraffin-embedded (FFPE) tissues, blood, semen, or other biological samples. Analysis of nucleic acids extracted using LCM from fresh or archived formalin-fixed paraffin-embedded (FFPE) sections are used to provide information about sample DNA (e.g., genotype) and RNA (e.g., gene expression), for example, as they relate to sample morphology and/or disease state. Using LCM, the biological sample may be examined using a microscope system and individual cells or group of cells may be selected and microdissected using one or more lasers. Through various LCM techniques, the selected sample may be separated from unwanted portions of the larger sample and transferred to a container, such as a well, vial, or tube, for preparing the selected sample for use in a downstream assay, experiment, or test, for instance, in a polymerase chain reaction (PCR) or sequencing assay or workflow.
One objective in many LCM applications is to provide a large yield of quality RNA from fresh or archived FFPE samples. The formalin fixing of an FFPE sample can induce molecular cross-linking within samples, which complicates retrieval of nucleic acids, as well as reduces their yield and quality. This affects the efficiency of downstream detection.
In addition to increasing quality and yield of selected samples from fresh or FFPE specimens, there exists a demand within the field to increase total throughput by reducing the amount of time needed to prepare samples for downstream testing. Existing FFPE RNA Isolation Kits recommend a lengthy overnight Proteinase K (ProK) digestion at 37 degrees Celsius for sample lysis, followed by several purification steps to retrieve RNA from LCM FFPE tissue prior to downstream analysis. This approach also requires sample transfer steps that can introduce additional variability in the yield and quality of the RNA. This multi-step workflow can be especially challenging for RNA isolation from low-input sample types, such as single cells and small populations of rare cells. Consequently, there is a need for an improved LCM FFPE lysis-based solution for extraction of RNA from limited LCM FFPE samples that maintains RNA integrity that increases yield for downstream applications, for example, for PCR or sequencing, such as next generation sequencing (NGS).
Therefore, new methods and systems that aid in the recovery of the high quality RNA from low input LCM-derived FFPE tissue material are highly desirable, as well as improved ways of processing such samples in a manner that reduces processing time and reduces or eliminates transfer of a selected sample.